G protein-coupled receptors are ubiquitous components of signal transduction systems. This project includes an assessment of the role of polyunsaturated phospholipids in modulating G protein-coupled signal transduction and an elucidation of the mechanism of action of ethanol in these systems. In these studies, the visual transduction pathway, a prototypical G protein-coupled system, is being used as a model system. The effect of alcohols, anesthetics, and lipid composition on: the kinetics and extent of formation of metarhodopsin II (MII), the G protein activating form of rhodopsin; MII/G protein complex formation; the rate of G protein activation; cGMP phosphodiesterase activation; and the GTPase activity of the G protein are being studied. We have shown that short chain alcohols, such as ethanol, promote MII formation, while longer chain alcohols, such as decanol, are inhibitory. Intermediate length alcohols show a smooth transition from excitatory to inhibitory. The magnitude of the observed effects are dependent on the phospholipid acyl chain composition. The effects of ethanol, acyl chain composition, and cholesterol are well correlated with changes in phospholipid acyl chain packing free volume, as characterized by the time-resolved fluorescence anisotropy behavior of the membrane probe, diphenylhexatriene. Our results strongly support a lipid-mediated mechanism of action for alcohols in modulating the activation of a G protein-coupled receptor. Our observations are best explained by a novel lipid packing model developed in this laboratory, in which the presence of polyunsaturated acyl chains, in mixed saturated-unsaturated and dipolyunsaturated acyl chain phospholipids, leads to the formation of lateral domains or clusters in the surface of the membrane. Many published studies demonstrate a requirement for higher than physiological ethanol doses to produce an observable response. We have shown that increasing the osmolality of the sample solution, so as to better simulate cytosolic conditions, increases the efficacy of ethanol, thereby reducing the ethanol requirement to achieve the same response observed in standard buffer solution.